1. Field of the Invention
Existing polymerase chain reaction (PCR) techniques have provided sensitive and specific means for detecting DNA-containing analytes. The need for time-consuming and expensive Southern blots for detecting very small amounts and a requirement for cell lysis and DNA extraction, however, have provided an incentive for simplification of the procedure. This invention relates to a two-step direct PCR method which combines biological and enzymatic amplification of PCR targets and simplifies the procedures for sample processing.
2. Description of the Prior Art
Conventional hybridization assay methods utilizing DNA as the means for determining the presence of a given analyte in a sample have been practiced for many years, but there have been problems and drawbacks associated with this method, such as a lack of sensitivity and specificity as well as the time required to carry out the assay. The advent of the polymerase chain reaction eliminated many of these concerns, e.g. sensitivity has been increased to the extent that the detection of a single cell in a sample is theoretically possible, but, while specificity and sensitivity have been increased, the method often requires time-consuming and expensive Southern blots in order to detect small numbers of cells.
In addition, some samples, such as soil (Bej et al. 1991. Appl. Environ. Microbiol. vol. 57, pp. 1013-1017), food products (Rossen et al. 1992. Int. J. Food Microbiol. vol. 17, pp. 37-45) and plant leaves (Demeke and Adams. 1992. Biotechniques, vol. 12, pp. 332-334; Rowhani et al. 1993. Phytopathology. vol. 83, pp. 749-7535), may also contain inhibitors of PCR (Prosen et al. 1993. Phytopathology. vol. 83, pp. 965-970; Rasmussen and Wulf. 1991. Detection of Pseudomonas syringae pv. pisi using PCR. pp. 369-376, and Tourte and Manceau. 1991. Direct detection of Pseudomonas syringae pathovar phaseolicola using the polymerase chain reaction (PCR). pp. 402-403 both in: Proceedings 4th International Working Group on Pseudomonas syringae Pathovars. Internat. Soc. Plant Pathology Committee on Phytopath. Bact. and Univ. di Firenze, Inst. di Pathologia, Florence, Italy) which are not easily removed by standard extraction procedures. For example, in studies (Prosen et al., supra) utilizing DNA from seed extracts containing relatively small numbers of pathogen, it was determined that cfu""s (on average 12 cfu/reaction) gave variable results in replicate amplifications. In contrast, tests with direct nested PCR carried out with culture aliquots without prior DNA extraction consistently gave strong PCR bands.
Another general problem with PCR techniques is the inability to differentiate between dead cells and live cells, which is important in many phytosanitary applications.
To overcome these problems we have developed a novel technique which can specifically detect viable cells in any environmental sample. To accomplish this goal, we have combined biological preamplification on a growth medium with direct PCR by introducing a plating step prior to PCR analysis. This modification, which we have named BIO-PCR, provides the benefit of biological amplification of PCR targets prior to enzymatic amplification. The technique significantly improves detection in samples with low levels of contamination and greatly reduces the detection of dead cells and/or free DNA.
In accordance with this development, it is the object of the invention to provide a novel assay method which combines a culture step and a direct polymerase chain reaction step resulting in a method having increased specificity and sensitivity and decreased interference from contaminants such as inhibitors and dead cells.